外泌体中X26nt调控CCND1促进肿瘤血管生成及其在胃癌液体活检中的应用

The diagnosis of early gastric cancer (EGC) is very crucial for the treatment of gastric cancer, which mainly depends on gastroscopy but lack of noninvasive screening indicators such as serum markers. The IRE1 α / XBP1 signaling pathway plays a key role in gene regulation and is associated with many diseases. At present, most XBP1 studies focus on the spliced XBP1(XBP1s), the activated form. Interestingly, our preliminary data showed X26nt, a by-product of XBP1 splicing, may promote cell proliferation via stabling CCND1 mRNA. What is more, we found that X26nt in patients’ serum exosomes with Ⅰ/Ⅱphase gastric cancer increased dramatically. Based on current progression and our preliminary data, we propose the hypothesis: X26nt promotes endothelial cells proliferation via up-regulation of CCND1 and changes vascular endothelial permeability, thereby promoting tumor angiogenesis and metastasis. This project intends to first identify the X26nt specific binding protein by mass spectrometry to clarify its secretion mechanism. Then we will construct the X26nt mutant gastric cancer cell line and proved that the X26nt derived from gastric cancer can promote the tumor growth. The effect of X26nt on proliferation and metastasis of tumor cells was further verified by mouse subcutaneous tumorigenesis model. Finally, we will evaluate the potential of X26nt as a serum marker of EGC by comparing the expression of X26nt in patients with different tumors and different stages of the gastric cancer.

早期胃癌的诊断对治疗和预后十分关键，但目前漏检率高，缺乏敏感特异的血清标志物。IRE1α/XBP1信号通路与众多疾病相关，我们前期研究表明XBP1选择性剪切对血管生成具有重要作用，最近发现XBP1剪切副产物X26nt在早期胃癌患者血清外泌体中异常升高，通过调控细胞周期蛋白CCND1促进细胞增殖。据此提出假说：在胃癌早期X26nt以外泌体形式分泌，通过上调CCND1促进内皮细胞增殖从而诱导肿瘤血管生成。本项目拟深入研究：1.鉴定X26nt在外泌体中特异性结合蛋白，明确其分泌机制；2.构建X26nt突变细胞株,明确胃癌细胞来源的外泌体X26nt对内皮细胞的诱导作用，并通过小鼠皮下成瘤模型进一步验证X26nt对肿瘤生长及转移的作用；3.检测不同肿瘤患者血清外泌体中X26nt的表达，与传统胃癌标志物做对比，综合评估X26nt在早期胃癌液体活检中的价值。

肿瘤微环境中的肿瘤相关内皮细胞 在胃癌发生发展中发挥重要的作用，然而其作用的详细机制至今尚不明确。外泌体作为肿瘤细胞和基质细胞间相互作用的重要介质，可以将多种功能性生物分子，包括蛋白质、编码RNA和非编码RNA（ncRNA）转移到细胞外间隙或生物液体中，从而介导细胞交流， 越来越多的研究表明其在胃癌进展中起到重要的调控作用。本研究的主要目的是探讨胃癌细胞来源外泌体在血管新生中的作用及机制研究。. 本课题采用1. qRT-PCR检测X26nt在不同肿瘤血清及血清外泌体中的表达水平，并通过胃癌组织芯片及荧光原位杂交实验（Fluorescence in situ hybridization，FISH）进一步检测X26nt在胃癌及癌旁组织中的表达；2. 分离并通过透射电镜、粒径分析、蛋白免疫印迹实验鉴定外泌体，EdU增殖实验、伤口愈合实验、Transwell迁移实验、跨内皮迁移实验和小管形成实验验证胃癌细胞外泌体中的X26nt对内皮细胞增殖、迁移和小管形成的影响；3. 使用TargetScan软件预测X26nt的靶基因，并用Luciferase实验加以验证，接着通过过表达和敲低X26nt验证其对靶基因表达的影响，进而证实胃癌细胞外泌体中的X26nt对内皮细胞增殖、迁移和小管形成影响的机制；4. 裸鼠皮下移植瘤模型验证X26nt对胃癌生长及血管生成的影响。. 重要结果：1. X26nt在胃癌血清及血清外泌体中高表达；2. X26nt在胃癌组织和胃癌细胞外泌体中高表达，并通过外泌体传递给内皮细胞，促进内皮细胞增殖、迁移和小管形成；3. X26nt通过降低VE-cadherin的表达、增加内皮细胞CCND1的表达，促进内皮细胞增殖、迁移和小管形成；4. 裸鼠皮下移植瘤实验证实X26nt促进胃癌的生长及血管新生。. 综上所述，胃癌细胞来源外泌体X26nt通过降低内皮细胞VE-cadherin的表达、增加CCND1的表达促进肿瘤的血管生成。